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WO1989001783A2 - Anticorps recombinant et procede - Google Patents

Anticorps recombinant et procede Download PDF

Info

Publication number
WO1989001783A2
WO1989001783A2 PCT/GB1988/000731 GB8800731W WO8901783A2 WO 1989001783 A2 WO1989001783 A2 WO 1989001783A2 GB 8800731 W GB8800731 W GB 8800731W WO 8901783 A2 WO8901783 A2 WO 8901783A2
Authority
WO
WIPO (PCT)
Prior art keywords
derived
ham
antibody
chimaeric
light chain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/GB1988/000731
Other languages
English (en)
Other versions
WO1989001783A3 (fr
Inventor
Mark William Bodmer
John Robert Adair
Nigel Richard Whittle
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Celltech R&D Ltd
Original Assignee
Celltech Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Celltech Ltd filed Critical Celltech Ltd
Priority to FI892137A priority Critical patent/FI892137A7/fi
Publication of WO1989001783A2 publication Critical patent/WO1989001783A2/fr
Publication of WO1989001783A3 publication Critical patent/WO1989001783A3/fr
Priority to DK216089A priority patent/DK216089D0/da
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3015—Breast
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S424/00—Drug, bio-affecting and body treating compositions
    • Y10S424/801—Drug, bio-affecting and body treating compositions involving antibody or fragment thereof produced by recombinant dna technology
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/867—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof involving immunoglobulin or antibody produced via recombinant dna technology

Definitions

  • the HAM of the present invention will be produced by recombinant DNA technology.
  • a process for producing the HAM of the first aspect of the invention which process comprises:
  • Figure 6 shows SDS-PAGE analysis in a reducing gel of immunoprecipitations from the supernatants of transfected COS-cells.
  • the DNA used for the transfection was as follows: Lane 1, mouse light chain alone; Lane 2, mouse light chain, mouse heavy chain; Lane 3, mouse light chain, chimaeric heavy chain; Lane 4, chimaeric light chain alone; Lane 5, chimaeric light chain, mouse heavy chain; and Lane 6, chimaeric light chain, chimaeric heavy chain.
  • the antibodies used for the immunoprecipitations were: Lanes 1-3, rabbit anti-mouse F(ab') 2 ; Lanes 4-6, rabbit anti-human F(ab') 2 ;
  • a genomic clone containing sequences coding for the human C ⁇ 4 region was isolated as a Hindlll fragment from the cosmid COS Ig8 (23) and then cloned via pAT153 into M13tg130 as an EcoRI-BamHI fragment to form pJA78.
  • an 18 mer oligonucleotide was synthesised and site specific mutagenesis was performed to convert a C residue to an A residue, thereby generating a novel Hindlll site at the start of the CH1 exon, to yield PJA91.
  • Site directed mutagenesis was performed (24) using EcoRI- and Bgll-cut M13mp18 to generate a gapped duplex with the relevant phage template. DNA was transformed into E.
  • Microtitre plates were coated with 0.25 ⁇ g per well of sheep or goat antibody reactive against either human or mouse specific epitopes on the heavy or light chains.
  • Supernatants or lysates from transfected COS cells were diluted 1:2 or 1:4 respectively in sample conjugate buffer containing 0.1M Tris-HCl pH 7.0, 0.1M sodium chloride, 0.02% Tween 20 and 0.2% casein. 100 ⁇ l of each diluted sample were added to each well and incubated for 1 hour at room temperature with gentle agitation.
  • DNA from plasmid EE6.cL.neo comprising the chimaeric light chain gene expressed from the HCMV promoter, was digested with Sall to generate linear molecules and then ethanol precipitated. The precipitate was resuspended in PBS and 40 ⁇ g of DNA was added to 10 7 CHO-K1 cells in buffer at 4°C. The DNA was introduced into the cells by electroporation, in which the cell suspension and DNA were treated with two pulses of 2000 volts. After electroporation the cells were returned to 4°C for 10 min prior to plating out at a density of 5 x 10 5 cells per 90mm Petri dish in DMEM growth medium containing supplements.
  • Resistant colonies were picked from the transformation plates using filter paper squares soaked in trypsin solution and transferred into individual wells of 24 well tissue culture plates.
  • the culture medium from the wells was assayed for the presence of chimaeric light chain using an ELISA assay and cell lines secreting light chain were identified. Lines producing light chain at levels between 100ng/ml and 16 ⁇ g/ml were identified. Those producing the highest amounts were cloned out by the limiting dilution method. One such clone, cL18, was used for subsequent studies.
  • Chimaeric B72.3 made in COS or CHO cells contains a proportion of material (10-20%) which fails to form an inter-heavy chain disulphide bridge but otherwise assembles correctly into 150kD molecules containing two heavy and two light chains. This material is present in antibody when secreted from the cell and co-purifies with antibody in which the inter-heavy chain disulphide bridges have formed. This appears to be a common property of human IgG4 molecules and occurs with all molecules of this type analysed to date, including a mouse-human IgG4 chimaeric anti-NP antibody and two different IgG4 myeloma proteins.
  • pJA79 is an M12tgl30 vector which contains the human IgG4 heavy chain gene modified so that the sequence from the first nucleotide after the last codon of the hinge exon to the last nucleotide of the CH3 domain inclusive has been removed by oligonucleotide directed site specific deletion.
  • the hinge and 3' untranslated region and part of the M13 sequence can be isolated as a 1.1 kbp Bglll fragment.
  • This fragment can be used to replace the analogous fragment in the full length B72.3/IgG4 chimaeric heavy chain gene clone pJA93 to give plasmid JA94 which therefore contains a chimaeric gene potentially capable of being expressed to produce a B72.3 IgG4 chimaeric F(ab') heavy chain protein.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Molécule d'anticorps humanisés (HAM) ayant une spécificité pour l'antigène TAG-72, et dotée d'un site de liaison d'antigènes dans lequel au moins les régions déterminantes complémentaires (CDRs) du domaine variable sont dérivées de l'anticorps monoclonal B72.3 (B72.3 MAb) de la souris, les parties dérivées d'immunoglobuline restantes de la HAM étant dérivées d'une immunoglobuline humaine, et procédé permettant sa production.
PCT/GB1988/000731 1987-09-04 1988-09-05 Anticorps recombinant et procede Ceased WO1989001783A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
FI892137A FI892137A7 (fi) 1987-09-04 1988-09-05 Yhdistelmävasta-aine ja menetelmä
DK216089A DK216089D0 (da) 1987-09-04 1989-05-03 Rekombinant antistof og fremgangsmaade til dets fremstilling

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB878720833A GB8720833D0 (en) 1987-09-04 1987-09-04 Recombinant dna product
GB8720833 1987-09-04

Publications (2)

Publication Number Publication Date
WO1989001783A2 true WO1989001783A2 (fr) 1989-03-09
WO1989001783A3 WO1989001783A3 (fr) 1989-04-20

Family

ID=10623277

Family Applications (3)

Application Number Title Priority Date Filing Date
PCT/GB1988/000730 Ceased WO1989001974A1 (fr) 1987-09-04 1988-09-05 Anticorps recombinant
PCT/GB1988/000731 Ceased WO1989001783A2 (fr) 1987-09-04 1988-09-05 Anticorps recombinant et procede
PCT/GB1988/000729 Ceased WO1989001782A1 (fr) 1987-09-04 1988-09-05 Anticorps recombinant

Family Applications Before (1)

Application Number Title Priority Date Filing Date
PCT/GB1988/000730 Ceased WO1989001974A1 (fr) 1987-09-04 1988-09-05 Anticorps recombinant

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/GB1988/000729 Ceased WO1989001782A1 (fr) 1987-09-04 1988-09-05 Anticorps recombinant

Country Status (11)

Country Link
US (1) US5219996A (fr)
EP (3) EP0348442B1 (fr)
JP (3) JPH02501191A (fr)
AT (2) ATE84973T1 (fr)
AU (3) AU611202B2 (fr)
CA (2) CA1337641C (fr)
DE (2) DE3888172T2 (fr)
DK (3) DK216089D0 (fr)
FI (3) FI892137A7 (fr)
GB (1) GB8720833D0 (fr)
WO (3) WO1989001974A1 (fr)

Cited By (28)

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WO1990007861A1 (fr) * 1988-12-28 1990-07-26 Protein Design Labs, Inc. IMMUNOGLOBULINES CHIMERIQUES SPECIFIQUES CONTRE LA PROTEINE TAC p55 DU RECEPTEUR D'IL-2
EP0403156A1 (fr) * 1989-06-07 1990-12-19 Genzyme Corporation Anticorps monoclonaux améliorés contre le récepteur alpha/bêta des cellules T humaines, leur production et leur utilisation
EP0438310A1 (fr) * 1990-01-19 1991-07-24 Merck & Co. Inc. Méthode de production d'immunoglobulines recombinantes
WO1991016928A1 (fr) * 1990-04-27 1991-11-14 Celltech Limited ANTICORPS ANTI-MOLECULE 1 d'ADHERENCE INTERCELLULAIRE CHIMERIQUE ADAPTES AU MODELE HUMAIN, PROCEDE DE PREPARATION ET D'UTILISATION
WO1991016927A1 (fr) * 1990-04-27 1991-11-14 Celltech Limited Anticorps anti-molecule 1 d'adherence intercellulaire a greffe de regions determinantes complementaires adaptes au modele humain, leur procede de preparation et d'utilisation
WO1992001059A1 (fr) * 1990-07-05 1992-01-23 Celltech Limited Anticorps diriges contre l'antigene carcino-embryonnaire (cea) et a greffe de zones determinant la complementarite (cdr) et production de ces anticorps
EP0365997A3 (fr) * 1988-10-19 1992-03-04 The Dow Chemical Company Famille d'anticorps modifiés à haute affinité pour le traitement du cancer
GB2276169A (en) * 1990-07-05 1994-09-21 Celltech Ltd Antibodies specific for carcinoembryonic antigen
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JPH02501191A (ja) 1990-04-26
EP0348442A1 (fr) 1990-01-03
FI892137A0 (fi) 1989-05-03
FI892137L (fi) 1989-05-03
DE3888172T2 (de) 1994-06-30
FI892135A7 (fi) 1989-05-03
JPH02501800A (ja) 1990-06-21
US5219996A (en) 1993-06-15
JPH02501190A (ja) 1990-04-26
FI892136A7 (fi) 1989-05-03
EP0347433B1 (fr) 1994-03-02
FI892135L (fi) 1989-05-03
FI892136L (fi) 1989-05-03
WO1989001782A1 (fr) 1989-03-09
JP2761012B2 (ja) 1998-06-04
ATE102254T1 (de) 1994-03-15
FI892135A0 (fi) 1989-05-03
AU614303B2 (en) 1991-08-29
FI892136A0 (fi) 1989-05-03
EP0348442B1 (fr) 1993-01-27
DK215889D0 (da) 1989-05-03
JP2675380B2 (ja) 1997-11-12
CA1337640C (fr) 1995-11-28
AU617367B2 (en) 1991-11-28
FI892137A7 (fi) 1989-05-03
AU2301688A (en) 1989-03-31
AU2301388A (en) 1989-03-31
ATE84973T1 (de) 1993-02-15
DK216089A (da) 1989-05-03
DK215889A (da) 1989-05-03
DE3877955T2 (de) 1993-07-22
DE3877955D1 (de) 1993-03-11
DK172630B1 (da) 1999-03-22
CA1337641C (fr) 1995-11-28
GB8720833D0 (en) 1987-10-14
AU2421888A (en) 1989-03-31
DK216089D0 (da) 1989-05-03
DK216189D0 (da) 1989-05-03
DK216189A (da) 1989-05-03
AU611202B2 (en) 1991-06-06
EP0347433A1 (fr) 1989-12-27
WO1989001974A1 (fr) 1989-03-09
EP0329755A1 (fr) 1989-08-30
DE3888172D1 (de) 1994-04-07
WO1989001783A3 (fr) 1989-04-20

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